1. Field of the Invention
The present invention relates generally to the fields of lipid biochemistry and liposomes. More particularly, the invention provides amphiphilic molecules that incorporate a hydrophilic material or polymer attached to two or more spatially distinct hydrophobic residues. On contact with water, these amphiphilic molecules display surface activity and self-assemble into multimolecular aggregates and liquid crystalline phases. The invention thus also provides liposomes of enhanced stability that incorporate such amphiphilic molecules, and methods of using these formulations in a variety of applications in the fields of drug delivery, nutrition, bio-diagnostics, cosmetics, blood products and related applications.
2. Description of Related Art
Amphiphilic molecules are so named because the structures contain hydrophilic and lipophilic (hydrophobic) parts. The molecules distribute across air-water and oil-water interfacial boundaries and display surface activity. In oil and water mixtures, these help form and stabilize emulsions and co-dissolve other materials. When dispersed in water at concentrations above critical solubility limits, these can be induced to self assemble into a variety of spatially ordered molecular aggregates including micelles and lamellar bilayers which can entrap other molecules in the lipid and/or the aqueous compartments of the aggregates. Amphiphile-containing emulsions, micelle, and lipid lamellar bilayer aggregates are important vehicles for parenteral delivery of therapeutic agents and nutrients.
Liposomes are spherical vesicles of self-closed hydrated bilayers of amphiphilic lipids surrounding a generally central inner aqueous phase core which can differ in composition from the extraliposomal aqueous medium (Bangham and Horne, 1964). The lipid chains may be liquid-crystalline or solid-like gel phases. Liposomes are colloidal particles ranging in diameter from 20 nm to 5000 nm. Depending on the size and the number of constituent lamellar layers, these are classified as small or large unilamellar vesicles, and as multilamellar vesicles. The multilamellar vesicles have additional water layers trapped adjacent to the hydrophilic ends (polar head groups) between the regular dual arrays of the lipophilic (hydrophobic) alkyl chains (fatty tails).
The lipid bilayer of the unilamellar vesicles is akin in composition and structure to the outer membrane of eukaryotic cells. The vesicle bilayer provides a significant controllable barrier to the movement of various molecules and ions between the inner aqueous core and the bulk aqueous phase surrounding the liposome (Bangham et al, 1965). This barrier function is paramount in many applications including drug delivery vehicles.
The need for and importance of a functional barrier is well illustrated by potential applications of liposomes in enzyme replacement therapies for inherited metabolic diseases, in other therapies using bioactive peptides and proteins, and in hemoglobin-based blood substitutes. The safety and efficacy of therapeutic/bioactive proteins depends upon their ability to overcome metabolic and transport barriers and reach the target site in a biologically active form. This in turn is dependent on the route for administration.
In general, exogenous proteins cause immunogenic and antigenic reactions and undergo rapid hydrolytic degradation in vivo. These problems can be partially alleviated by modification of the protein by covalent conjugation to a biocompatible hydrophilic polymer such as a monofunctional polyethyleneglycol (PEG). As an example, adenosine deaminase conjugated to .omega.-methyl-polyethyleneglycol (MePEG) of average molecular weight 5000 shows lower immunogenicity and antigenicity, and prolonged blood circulation half-life. However, although the conjugate has seen some clinical use, it has relatively low enzyme activity (Beauchamp et al., 1983). Despite the loss of bioactivity on covalent modification of proteins that is known to occur generally, other MePEG-inked proteins, including the immunoregulatory cytokine interleukin-2, and the oxygen transporter hemoglobin, are being investigated for ultimate use in vivo.
The extra effort needed to develop reasonably active conjugates for every enzyme contemplated for in vivo is a significant limitation in this field. This could be avoided if an encapsulation process applicable to all unmodified enzymes could be developed, but this has yet to be achieved. Potential systemic and transdermal delivery systems for unmodified bioactive proteins, such as entrapment in biodegradable microspheres fabricated from poly(lactide-co-glycolide) and other polymers, are being investigated (Gombotz and Pettit, 1995). Liposomal systems have also been proposed and, although these offer the advantage that they are capable of self-assembly (Gregoriadis, 1988), they currently suffer from certain drawbacks.
Liposomes are normally prepared from natural phospholipids and synthetic analogues such as the electrical charge neutral zwitterionic phosphatidylcholines. Minor proportions of anionic phospholipids, such as phosphatidylglycerols, are added to generate a net negative surface charge for colloid stabilization. The lipid chains in the bilayer may be present as crystalline or mesophase (liquid-crystalline) states. The type of mesophase controls the physical integrity of the liposomes in vitro and in vivo, and liposomes with gel phase bilayers are more stable in blood than those with liquid-crystalline bilayers.
For liposomes administered parenterally, the blood circulation half-life, distribution and disposition in organs and tissues is correlated strongly with the diameter and the surface properties of the liposomes. Most liposomes are rapidly taken up by the phagocytic cells of the reticuloendothelial system (RES), the circulating mononuclear phagocytic cells and those located in the liver and spleen, and their blood circulation half-lives are short (a few minutes). This uptake is generally mediated by binding of plasma proteins (opsins) to the liposomal surface. The non-specific "scavenger receptor" that recognizes negative charges in large arrays may be involved also (Nishikawa et al., 1990). Liposomes smaller in diameter than the average diameter of the fenestrae in the blood capillaries leak out. The average diameter of the fenestrae in the imperfectly formed sinusoids in rapidly growing tumors is larger than in normal tissues and therefore liposomes smaller than about 100 nm in diameter migrate into tumors.
The above two proclivities provide the basis for targeting liposome-encapsulated drugs to liver and tumors respectively. A primary requirement for targeting therapies for metabolic disorders to other organs and tissues is that liposomes be able to evade uptake as above and have long blood circulation half-life. The inclusion of ganglioside G.sub.M1, a natural glycolipid with terminal sialic acid residue, as a minor envelope component improves circulation life, presumably because of changes in the liposomal surface characteristic (Allen and Chonn, 1987). However, this has yet to yield sufficiently beneficial results.
Recently, certain .omega.-methyl-polyethyleneglycol-conjugated anionic lipids have been developed, notably .omega.-Me-PEG-phosphatidylethanolamines (MePEG-PE), and used as envelope components at about 5 mole % of total lipid. The resulting liposomes display pendant MePEG residues on the outer lipid envelope surface, and these are considered to act as steric barriers to opsin attachment and RES uptake (Lasic et al., 1991; Needham et al., 1992; Woodle and lasic, 1992). Therefore, these liposomes are called sterically stabilized liposomes. The degree of polymerization and surface density of the MePEG, and anionic charge are important parameters for liposome stability. However, even with optimum parameters, these sterically stabilized liposomes only have a blood circulation half-life of between about 12 and about 48 hours, as compared to the blood circulation half-life of red blood cells of 28 days.
Antibodies and receptor-specific ligands have also been tethered to the surface of sterically stabilized liposomes for inducing targeted delivery to specific tissues. Counterproductively, the pendant MePEG chains in these mixed surface ligand type liposomes not only prevent uptake by the RES but also hinder the approach and binding of liposome-surface-linked antibodies to the target tissue receptors. Therefore, the art is, at best, ambivalent about their potential for targeted delivery to specific tissues. For instance, according to an authoritative comment "the remaining problems of accessibility of a particular tissue and cells as well as overlooked severity of triggering an immune response to the host organism by antibody or lectin-coated liposomes makes this goal rather remote at present" (Lasic and Barenholz, 1996).
In an attempt to generate liposomes of improved stability, two broad trends are discernible in the current research literature. There is continuing interest in (i) optimizing existing liposome types, and (ii) in developing alternative systems. Optimization is being attempted by the development of alternatives lipid conjugates of MePEG. The preparation and applications of diacylglycerol and cholesterol in place of PE, and for the MePEG-PE series, the investigation of different spacers and chemistries for conjugating MePEG to PE have been described (Parr et al., 1994; Zalipsky, 1995). However, these have still not countered the prevailing pessimism explained above. Alternatively, various hydrophilic polymers other than MePEG have been conjugated to PE, but only poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline) conjugates have afforded even the minimum protection from hepatosplenic uptake seen with MePEG-PE (Woodle et al., 1994).
Data on the retention and chemical stability of MePEG-lipid conjugates incorporated into unilamellar vesicles are available. Many MePEG-lipid stabilized liposomes gave very little improvement in circulation half-life, and this was traced to the rapid removal of the hydrophilic coating. The latter is attributed to the loss of the intact MePEG-lipid from the liposomal membrane. Significant chemical breakdown of MePEG-lipid (MePEG-PE) occurs, especially after its detachment from the liposome body. Finally, the loss of hydrophilic coating precedes liposome clearance (Parr et al., 1994).
Focusing on the properties inherent in the MePEG-lipid conjugate structure, the present inventor has reevaluated these data in terms of relative propensity of the conjugate for retention in the liposome bilayer and proclivity for migration into the extraliposomal fluid, and, the mole % of MePEG-lipid in the bilayer phospholipids needed for complete coverage with an adequate depth of MePEG chains. For representative phospholipid structures, conjugates with relatively short MePEG chains tend to stay in the bilayer but provide inadequate surface barrier. Conjugates with longer chains tend to migrate into the surrounding aqueous medium, particularly at high concentrations which provide adequate surface barrier. The inventor realized that the balance of these opposing influences/conflicting constraints cannot be improved further without a radical departure from the structures typified by MePEG-lipid. In particular, these efforts cannot provide the minimum one order of magnitude increase in half-life needed for encapsulated functional proteins such as hemoglobin for blood substitutes.